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Hi Matthew, TEV is probably the least robust protease among those commonly used for tag removal. Here¡¯s a common unit definition of TEV. One unit (corresponding to 0.1 ug TurboTEV) cleaves ¡Ý85% of 3 ¦Ìg control substrate in 1 hour at 30C. You need to use really a lot of TEV. Information collected from our customers shows large variation in quality of home-made TEV proteases. Some used 1:5 w:w ratio. Adding a little bit EDTA to Ni pool helps TEV digestion. You can find a general protocol at http://www.accelagen.com/TurboTEV-protocol.htm. Cheers, Chun Accelagen _____ From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Matthew Merski Sent: Monday, May 24, 2010 9:28 AM To: [log in to unmask] Subject: [ccp4bb] TEV cleavage problems Hello all, I am working with a protein that is expressed as with an N-terminal domain that is normally cleaved for activation of the protein (and crystallization). For in vitro reasons I've needed to switch the normal site to a TEV site. However, even though the TEV site is in the same place as the original proteolytic site, I have been unable to get cleavage despite using a lot of TEV at 37 C, pH 8.0. Has anyone been able to overcome a similar problem? Matthew Merski Post-doctoral researcher UCSF